1. Field of the Invention
This invention relates generally to the field of optical illumination and detection of biological activity in cells, organs or other samples, and more specifically to a method and apparatus for observing virtually instantaneously, the activity of a sample by illuminating the sample and measuring the light emitted from the sample.
2. Discussion of the Prior Art
Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms of a sample move from one energy level to another. In biological research, an observation in the changes of the optical absorption and emission of light provide valuable scientific information of what is occurring in the cell, organ or sample, for example, the progress of an ongoing chemical reaction.
Chemical analysis using absorption spectroscopy allows for the determination of concentrations of specific components, to assay reactions and to identify individual compounds while fluorescence is a physical phenomena based upon the ability of some substances to absorb and subsequently emit radiation. The emitted radiation generally has a lower energy level and a longer wavelength than the absorbed radiation which is used to excite the sample. Furthermore, the absorption of the incident light is wavelength dependent. Thus, a sample will only fluoresce when the excitation wavelength of the incident light falls within the excitation band for the substance at that particular time. The phase relationship between variations in the excitation or incident light and the light emitted from the sample is very important in observing changes in a substance as an event or reaction occurs.
One of the deficiencies of the prior art has been the lack of ability to faithfully observe ongoing changes in a sample as a reaction occurs, when the reaction requires detection at several wavelengths of light. With the present state of the art, it is often required in analytical procedures that reactions or assays be monitored at more than one wavelength. For observation systems that can change wavelengths in a little as one second, or theoretically, in {fraction (1/30)}th of a second, there is high likelihood that a change in a sample occurring in {fraction (1/100)}th or {fraction (1/1000)}th of a second will not be detected. Also, in some analytic procedures, it is a requirement to change the wavelength of the excitation and emission light to permit detection at several wavelengths in order for a meaningful measurement to be made.
In newer approaches in the detection of events that occur in cells or other biologically related specimens, meaningful data must be obtained from the compilation of measurements made at more than one excitation and/or emission wavelength. As such, the speed of detection by the detecting device (PMT) is as important as the ability of the instrument to change the excitation and/or detect emission wavelength in a rapid fashion in-between or during the detection events. An instrument that has the ability to rapidly change excitation wavelengths and to rapidly detect a plurality and emission wavelengths in a short period of time, and synchronize events based on detection of light emitted by the sample, can monitor events that occur in the specimen with higher fidelity. Examples of these approaches and procedures for detection are the use of readily available fluorescent chemicals that exhibit spectrum-shifting properties that are dependent on their environment. Fura-2 and indo-1 are two typical chemicals used for the determination of calcium concentration that exhibit spectrum-shifting properties in certain environments.
One device for excitation/emissions measurements invented by Barlow et al. and described in U.S. Pat. No. 5,422,730, sets forth a system which permits for detection by a CCD camera of ongoing reactions in a particular sample. The device set forth below by Barlow et al. uses a pair of filter wheels to select a plurality of filters that transmit pre-selected light wavelengths, but the selection of wavelengths by the two filter wheels are performed in a sequential fashion. The limitations of this device are that it permits detection of events at only one excitation and one emission wavelength. The sequential selection of wavelengths by the filter wheels detrimentally slows the response time of the instrument to obtain meaningful data if more than one excitation or more than one emission wavelength measurement is required for meaningful information.
Other devices for observing reactions in samples can excite a sample using two different wavelengths simultaneously or can measure two different wavelengths at the same time by utilizing beam splitting devices. Some of the more advanced equipment is capable of measuring four different wavelengths emitted by a sample by use of vibrating mirrors, choppers or dichroic mirrors. Each of these devices has the ability to select an excitation or emission wavelength in a rapid fashion, but they do comprise a system in which the excitation and emission wavelengths are changed simultaneously and are synchronized to the detection of light intensity.
There is lacking in the biological field a photometer that is capable of exciting a cell or sample with a plurality of wavelengths, typically more than four, and virtually instantaneous observing the ongoing reaction by analyzing a plurality of wavelengths, typically more than four, emitted by the cell, organ or sample, either in-vitro or in-vivo, that does not interfere with the activity of the cell, organ or sample, and which is inexpensive and easy to operate. While there are instruments available that can make measurements quickly, their ability to do so at multiple wavelengths is limited. Typically, these instruments utilize spinning or vibrating mirrors to change either the excitation or the emission wavelengths (but not both) quickly. Conversely, instruments capable of exciting samples and taking measurements at multiple wavelengths are slow. There is a need for an instrument that can accomplish both excitation and detection quickly.
Accordingly, an advantage of the present invention is the capability of exciting a cell, organ or sample with a plurality of wavelengths and measuring a plurality of wavelengths emitted by the cell, organ or sample. The measurements are performed hundreds of times per second and may be performed thousands of times per second so that rapidly occurring changes can be observed. Despite the rapid number of measurements made by the device of the present invention, the narrow wavelength band of incident light is always synchronized with a narrow wavelength of emitted light.
Another advantage of the present invention the equipment used for florescent illumination and detection is relatively inexpensive to manufacture and does not require a high degree of skill to use.
Newer analytical procedures require the measurement of light intensity at more than one excitation or emission wavelength in order to get meaningful data. Measurement at one wavelength is not meaningful in the absence of another related measurement at a different wavelength. The results of both measurements are required for a calculation in the assay procedure. An advantage of the present invention is that it provides the capability to rapidly excite an assay or sample at a number of preselected wavelengths and to measure the emission from the assay at a number of preselected wavelengths while correlating the excitation wavelengths to the emissions wavelengths.
Meaningful data from an assay requires the capability to detect and measure a plurality of wavelengths of light emitted from an assay in a very short time frame. An advantage of the present invention is that it has the capability to both excite a sample or assay and detect the emissions from an assay at a plurality of wavelengths in a very short time frame so that meaningful multi-wavelength measurements can be made and recorded.
Still another advantage of the present invention is that it can readily be adapted for use with an optical microscope for real time viewing, with a CCD camera for real time viewing and recording or with a computer for digitizing, analyzing and recording the output from the sample.
In its broadest embodiment, the present invention is a multichannel system, each channel being independent yet synchronized with the other channel. The multichannel system can be used effectively for high speed synchronization of light from a light source incident on a cell, tissue, organ or other biological sample and light emitted from the cell, tissue, organ or other biological sample as a result of biological or chemical activity occurring in it. The multichannel system is comprised of a means for controlling operation of the system by issuing instructions. This central control unit is typically a CPU or central processing unit that is capable of executing a program that includes a plurality of sets of instructions or programs. Each program or series of instructions sets forth the operations that are to be performed by the system so that the desired measurements can be made. The operator preselects the program that is to be used through the central control unit. The central control unit is a device having a programmable memory which may optionally be connected to another device having a programmable memory, such as a programmable logic device (PLD) that is in communication with the central control unit. The device has a programmable memory for receiving instructions from the central control unit to execute a pre-selected program that has been preprogrammed into the device. The device also receives instructions from other hardware, these additional instructions being required for execution of the preselected program. The multichannel system also includes at least two independent integrated circuit devices capable of being programmed, such as preprogrammed motor controllers, that receive instructions from the devices and information from other hardware. Instrumental in the synchronization of the system is a clock that provides a timing signal to initiate operation of the program in the device having a programmable memory, such as the PLD, which in turn provides additional signals to permit synchronization of the operations of the motor controllers. Upon receiving a signal from the central control unit, the programmable device initiates operation of the preselected program in its memory that includes transmitting a signal simultaneously to each of the controllers to initiate their preprogrammed series of instructions. Connected to each motor controller is a motor driver, which receives power from any conventional power source. The motor drivers operate in response to signals received from their respective motor controllers. The motor drivers drive motors to which they are connected, each motor having a motor shaft. Connected to each motor shaft, so that they rotate with the shaft, is an encoder and a filter wheel, each filter wheel including a plurality of filter positions for holding filters. Each controller is capable of resolving the position of the encoder, and hence the filter wheel with a high degree of accuracy as they rotate on the motor shaft. The ability of the controllers to resolve their position is based on the ability of encoders to produce a plurality of pulses, and their ability to index these pulses to an initial position. Because each encoder is connected to the common shaft of the motor, resolution of the encoder position simultaneously resolves the position of the motor shaft and the corresponding filter wheel. Each controller resolves the encoder position with respect to the initial index pulse provided to the individual controller by the encoder. Each controller, in executing its set of instructions, receives signals from its respective encoder showing the actual position of the encoder with respect to its initial pulse and compares this actual position to a calculated position. If the actual and calculated positions do not correspond, the controller sends additional signals to the motor driver to speed up or slow down the motor so that the encoder position can be changed in the appropriate fashion. In this way the calculated position and the actual position of the encoder are brought into correspondence. Of course, because the encoder and the filter wheel are rotating on the same shaft, the position of the filter wheel is also known.
The programmable device first synchronizes the initial position of the filter wheels and encoders by providing a signal that sets them to the same stationary index position. In response to a command from the central control unit, the programmable device issues an instruction to each of the motor controllers to initiate spinning of the motors at the same constant speed at the same time based on the clock, using a preprogrammed sequence in each controller. The programmable device may optionally issue the instruction simultaneously to the controllers to initiate the program immediately. The controllers respond and enter a program loop in which they determine the actual position of the encoders at a given time and compare these actual positions to calculated positions of the encoders, the calculated positions being a function of motor speed and time. If the actual position is lagging the calculated position, the controller sends a signal to the motor driver to increase the speed of the motor, or if ahead of the calculated position, to decrease the speed of motor. Since both controllers are operated from the same clock, and are instructed to initiate operation simultaneously at the same speed, they both will calculate the same calculated position for their respective encoders in each cycle of their program loops. Adjustments by the controllers to the actual positions of their respective encoders will be based on the deviation of the encoders from their calculated positions. Because the calculated position of each encoder is the same, based on simultaneous execution of the same program within the programmable memory of the encoder, the controllers synchronize the positions of the encoders. These adjustments to the respective encoders are performed independently by each controller. This allows the two controllers to operate independently, while keeping the two encoders in synchronization. Because the filter wheels are fixed in relation to the encoders, operation of the filter wheels is also synchronized.
The synchronous yet independent operation of the filter wheels in this multichannel system provides the advancement in the state of the art. Since each filter wheel includes a plurality of filters, each of which transmit a narrow band of wavelengths, synchronization of a filter wheel having preselected filters to transmit a first narrow band of light of first preselected wavelengths to excite a sample, with a second filter wheel having preselected filters to receive a second narrow band of light of second preselected wavelengths permits rapid observations and measurement of emissions from the same sample when the emissions correspond to changes in the sample as a result of biological or chemical activity.
Other features and advantages of the present invention will be apparent from the following more detailed description of the preferred embodiment, taken in conjunction with the accompanying drawings which illustrate, by way of example, the principles of the invention.